Analysis of Recombinant Human ADP-Ribosylation Factors by Reversed-Phase High-Performance Liquid Chromatography and Electrospray Mass Spectrometry

Two complementary approaches utilizing reverse-phase high-performance liquid chromatography and liquid chromatography/mass spectrometry were developed to analyze recombinantly produced Group I and Group II human ADP-ribosylation factors (ARFs). We observe that the NH2termini of Group II ARFs (ARF4 and ARF5) are efficiently processed by removal of the initiating methionine. In contrast, the NH2termini of Group I ARFs (ARF1 and ARF3), although fully deformylated, undergo only partial methionine cleavage. This result is unexpected as ARFs are canonical substrates for methionine processing in both bacterial and eukaryotic systems, but it may explain the difficulties encountered by many researchers attempting to produce myristoylated ARFs inEscherichia coli.Additionally, we observe that a significant fraction of purified ARF4 contains a modification which we demonstrate to be consistent with mono-glutathionation. Both methionine retention and glutathione modification may impact ARF function and the methods presented here should be employed to determine the quality of recombinant ARFs.